RESUMO
BACKGROUND: Rhizobium-legume symbiosis is a specific, coordinated interaction that results in the formation of a root nodule, where biological nitrogen fixation occurs. NADPH oxidases, or Respiratory Burst Oxidase Homologs (RBOHs) in plants, are enzymes that generate superoxide (O2 â¢-). Superoxide produces other reactive oxygen species (ROS); these ROS regulate different stages of mutualistic interactions. For example, changes in ROS levels are thought to induce ROS scavenging, cell wall remodeling, and changes in phytohormone homeostasis during symbiotic interactions. In common bean (Phaseolus vulgaris), PvRbohB plays a key role in the early stages of nodulation. RESULTS: In this study, to explore the role of PvRbohB in root nodule symbiosis, we analyzed transcriptomic data from the roots of common bean under control conditions (transgenic roots without construction) and roots with downregulated expression of PvRbohB (by RNA interference) non-inoculated and inoculated with R. tropici. Our results suggest that ROS produced by PvRBOHB play a central role in infection thread formation and nodule organogenesis through crosstalk with flavonoids, carbon metabolism, cell cycle regulation, and the plant hormones auxin and cytokinin during the early stages of this process. CONCLUSIONS: Our findings provide important insight into the multiple roles of ROS in regulating rhizobia-legume symbiosis.
Assuntos
Carbono/metabolismo , Ciclo Celular , NADPH Oxidases/metabolismo , Phaseolus/enzimologia , Nodulação , Raízes de Plantas/enzimologia , Simbiose/fisiologia , Phaseolus/genética , Phaseolus/microbiologia , Raízes de Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Rhizobium/fisiologia , TranscriptomaRESUMO
Two microcystins, MC-LR and [D-Leu1]MC-LR, present in La Plata Basin blooms, are differentiated by substitution of D-Alanine for D-Leucine at position 1. Our objective was to evaluate acute toxicity of [D-Leu1]MC-LR and MC-LR in mice (N:NIH Swiss) and beans (Phaseolus vulgaris). We observed variations in [D-Leu1]MC-LR lethal doses with respect to those reported for MC-LR (100 µg/kg), with an increased liver/body weight ratio and intrahepatic hemorrhages in mice exposed to 50-200 µg [D-Leu1]MC-LR/kg and slight steatosis after a single 25 µg [D-Leu1]MC-LR/kg i.p. dose. Our study in the plant model showed alterations in germination, development, morphology and TBARs levels after a single contact with the toxins during imbibition (3.5 and 15 µg/mL), those treated with [D-Leu1]MC-LR being more affected than those treated with the same concentration of MC-LR. Protein phosphatase 1 (PP1) IC50 values were 40.6 nM and 5.3 nM for [D-Leu1]MC-LR and MC-LR, respectively. However, the total phosphatase activity test in root homogenate showed 60% inhibition for [D-Leu1]MC-LR and 12% for MC-LR. In mouse liver homogenate, 50% inhibition was observed for [D-Leu1]MC-LR and 40% for MC-LR. Our findings indicate the need for further research into [D-Leu1]MC-LR toxicity since together with oxidative stress, the possible inhibition of other phosphatases could explain the differences detected in the potency of the two toxins.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Inibidores Enzimáticos/toxicidade , Fígado/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Microcistinas/toxicidade , Phaseolus/efeitos dos fármacos , Proteínas de Plantas/antagonistas & inibidores , Proteína Fosfatase 1/antagonistas & inibidores , Animais , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Phaseolus/enzimologia , Proteínas de Plantas/metabolismo , Proteína Fosfatase 1/metabolismoRESUMO
[D-Leu1]MC-LR and MC-LR, two microcystins differing in one amino acid, constitute a sanitary and environmental problem owing to their frequent and concomitant presence in water bodies of the Americas and their association with human intoxication during recreational exposure to cyanobacterial bloom. Present in reservoirs used for irrigation as well, they can generate problems in the development of crops such as Phaseolus vulgaris, of nutritional and economic interest to the region. Although numerous works address the toxic effects of MC-LR, information on the toxicity of [D-Leu1]MC-LR is limited. Our objective was to study the toxic effects of [D-Leu1]MC-LR and MC-LR (3.5 µg/ml) on P. vulgaris after a single contact at the imbibition stage. Our findings indicate that 10 days post treatment, [D-Leu1]MC-LR generates morphological and physiological alterations more pronounced than those caused by MC-LR. In addition to the alterations produced by [D-Leu1]MC-LR in the development of seedlings and the structure of the leaves, roots and stems, we also found alterations in leaf stomatal density and conductivity, a longer delay in the phototropic response and a decrease in the maximum curvature angles achieved with respect to that observed for MC-LR. Our findings indicate that these alterations are linked to the greater inhibition of phosphatase activity generated by [D-Leu1]MC-LR, rather than to oxidative damage. We observed that 30 days after treatment with MC-LR, plants presented better development and recovery than those treated with [D-Leu1]MC-LR. Further studies are required on [D-Leu1]MC-LR and MC-LR toxicity and their underlying mechanisms of action.
Assuntos
Toxinas Marinhas/toxicidade , Microcistinas/toxicidade , Phaseolus/efeitos dos fármacos , Processos Fototróficos/efeitos dos fármacos , Desenvolvimento Vegetal/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Phaseolus/enzimologia , Phaseolus/crescimento & desenvolvimento , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Reactive oxygen species (ROS) are generated by NADPH oxidases known as respiratory burst oxidase homologs (RBOHs) in plants. ROS regulate various cellular processes, including the mutualistic interactions between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. Rboh is a multigene family comprising nine members (RbohA-I) in common bean (Phaseolus vulgaris). The RNA interference-mediated silencing of RbohB (PvRbohB-RNAi) in this species diminished its ROS production and greatly impaired nodulation. By contrast, the PvRbohB-RNAi transgenic roots showed early hyphal root colonization with enlarged fungal hypopodia; therefore, we proposed that PvRbohB positively regulates rhizobial infection (Rhizobium tropici) and inhibits AM colonization by Rhizophagus irregularis in P. vulgaris. RESULTS: To corroborate this hypothesis, an RNA-Seq transcriptomic analysis was performed to identify the differentially expressed genes in the PvRbohB-RNAi roots inoculated with Rhizobium tropici or Rhizophagus irregularis. We found that, in the early stages, root nodule symbioses generated larger changes of the transcriptome than did AM symbioses in P. vulgaris. Genes related to ROS homeostasis and cell wall flexibility were markedly upregulated in the early stages of rhizobial colonization, but not during AM colonization. Compared with AM colonization, the rhizobia induced the expression of a greater number of genes encoding enzymes involved in the metabolism of auxins, cytokinins, and ethylene, which were typically repressed in the PvRbohB-RNAi roots. CONCLUSIONS: Our research provides substantial insights into the genetic interaction networks in the early stages of rhizobia and AM symbioses with P. vulgaris, as well as the differential roles that RbohB plays in processes related to ROS scavenging, cell wall remodeling, and phytohormone homeostasis during nodulation and mycorrhization in this legume.
Assuntos
Perfilação da Expressão Gênica , Glomeromycota/fisiologia , NADPH Oxidases/genética , Phaseolus/genética , Phaseolus/microbiologia , Raízes de Plantas/genética , Rhizobium tropici/fisiologia , Parede Celular/metabolismo , Phaseolus/citologia , Phaseolus/enzimologia , Raízes de Plantas/microbiologia , Transdução de Sinais/genética , SimbioseRESUMO
Considering that the mechanisms for phosphite-afforded disease control remain elusive, this study investigated whether zinc (Zn) and copper (Cu) phosphites could possible potentiate common bean resistance to white mold, caused by Sclerotinia sclerotiorum, through the stimulation of biochemical defence responses. Lesion area and disease severity were decreased by phosphites spray, but Zn phosphite outcompeted Cu phosphite. Histopathological observations revealed fewer fungal hyphae and less collapse of the mesophyll cells in the Zn and Cu phosphite-sprayed plants compared to water-sprayed ones. The S. sclerotiorum-triggered accumulation of reactive oxygen species, oxalic acid (a fungal secreted toxin) and malondialdehyde (an indicator of cellular damage) were constrained as a result of Zn and Cu phosphites spray. Activities of antioxidant enzymes (superoxide dismutase, peroxidase, ascorbate peroxidase and glutathione-S-transferase at 12â¯h after inoculation (hai) and catalase at 60 and 84 hai) were higher for Zn and Cu phosphites-sprayed plants than for water-sprayed ones. Activities of defence-related enzymes chitinase (CHI) at 12 hai, ß-1,3-glucanase (GLU) and polyphenoloxidase (PPO) were higher at 12-84 hai for Zn, and Cu phosphites sprayed plants, phenylalanine ammonia-lyase at 36-84 hai for the Zn phosphite sprayed ones, CHI at 12-36 hai, GLU at 12-60 hai, PPO at 36 hai and PAL and lipoxygenase at 12 hai for the Cu phosphite sprayed ones upon inoculation with S. sclerotiorum relative to their water-sprayed counterparts. Concentrations of total soluble phenols and lignin-thioglycolic acid derivatives were not affected by Cu phosphite spray on infected plants but were higher and lower, respectively, for Zn phosphite sprayed plants at 60 hai compared to water-sprayed ones. Taken together, the findings from the present study shed light on the biochemical defence mechanisms involved in the Zn and Cu phosphites-mediated suppression of white mold in common bean.
Assuntos
Ascomicetos/fisiologia , Phaseolus/microbiologia , Fosfitos/farmacologia , Análise de Variância , Antioxidantes/metabolismo , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Malondialdeído/metabolismo , Phaseolus/citologia , Phaseolus/efeitos dos fármacos , Phaseolus/enzimologia , Fenóis/metabolismo , Doenças das Plantas/microbiologia , Análise de Componente Principal , Solubilidade , Superóxidos/metabolismo , Tioglicolatos/metabolismoRESUMO
Dry bean (Phaseolus vulgaris L.) is an important economic crop in Brazil but its yield can be significantly reduced by white mold, a disease caused by Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic, highly destructive, and non-host-specific fungus. This fungus secretes numerous cell wall-degrading enzymes such as polygalacturonases, exo-ß-1,3-glucanases, xylanases, and cellulases that have been detected during the early stages of infection. In this study, the activities of these enzymes were detected in all carbon sources tested (citrus pectin, cell wall extract from P. vulgaris, carboxymethyl cellulose, and glucose), but the highest levels were found when using citrus pectin and cell wall extract from P. vulgaris. Regardless of the carbon source, pH decreased throughout the culture time. During pathogenesis in dry bean stems, increased enzyme activities were also observed. Reverse transcriptase-polymerase chain reaction experiments showed that the induction of polygalacturonases (sspg1, sspg3, sspg5, sspg6, and sspg7), exo-ß-1,3-glucanases, and endo-ß-1-4-glucanase in S. sclerotiorum occurred during the early stages of colonization.
Assuntos
Ascomicetos/enzimologia , Celulase/genética , Glucana 1,3-beta-Glucosidase/genética , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Poligalacturonase/genética , Sequência de Bases , Parede Celular , Dados de Sequência Molecular , Pectinas , Phaseolus/enzimologia , Extratos Vegetais/metabolismoRESUMO
Symbiotic legume-rhizobia relationship leads to the formation of nitrogen-fixing nodules. Successful nodulation depends on the expression and cross-talk of a batttery of genes, among them SymRK (symbiosis receptor-like kinase), a leucine-rich repeat receptor-like kinase. SymRK is required for the rhizobia invasion of root hairs, as well as for the infection thread and symbiosome formation. Using immunolocalization and downregulation strategies we have recently provided evidence of a new function of PvSymRK in nodulation. We have found that a tight regulation of PvSymRK expression is required for the accurate development of the vascular bundle system in Phaseolus vulgaris nodules.
Assuntos
Phaseolus/enzimologia , Proteínas de Plantas/metabolismo , Feixe Vascular de Plantas/enzimologia , Proteínas Quinases/metabolismo , Nódulos Radiculares de Plantas/enzimologia , Simbiose , Proteínas de Plantas/química , Proteínas Quinases/químicaRESUMO
Phosphorus is an essential element for all living cells, but its availability is often limiting in the soil. Plants have adapted to such limitation and respond to phosphorus deficiency. The soluble inorganic pyrophosphatases (PPase; EC 3.6.1.1) recycle the pyrophosphate produced by many biosynthetic reactions, and may play a role in the plant adaptation to phosphorus deficiency. In this work, three PPase mRNAs were identified from the Phaseolus vulgaris EST international database and their sequences were corroborated and completed using 3'RACE. After design and validation of the appropriate oligonucleotide primers, the PPase mRNA expression was measured by qRT-PCR in leaves, stems, and roots of bean plants grown with 1mM phosphate or under phosphate starvation. The plant tissues were classified according to their position on the plant, and some physiological signs of stress were recorded. qRT-PCR revealed changes in mRNA expression, but not for all isozymes under analysis, and not for all tissues. In addition, changes in the activity of some PPases were observed in zymograms. Our data are consistent with an important role for pyrophosphate in the adaptation of the plant to phosphate starvation.
Assuntos
Adaptação Fisiológica , Expressão Gênica , Pirofosfatase Inorgânica/metabolismo , Phaseolus/enzimologia , Fosfatos/metabolismo , Fósforo/deficiência , Estresse Fisiológico , Sequência de Bases , Primers do DNA , Difosfatos/metabolismo , Regulação da Expressão Gênica de Plantas , Pirofosfatase Inorgânica/genética , Isoenzimas , Phaseolus/genética , Phaseolus/metabolismo , Fósforo/metabolismo , Estruturas Vegetais , RNA Mensageiro/metabolismo , Solo , SolubilidadeRESUMO
The symbiotic interaction of legumes and rhizobia results in the formation of nitrogen-fixing nodules. Nodulation depends on the finely coordinated expression of a battery of genes involved in the infection and the organogenesis processes. After Nod factor perception, symbiosis receptor kinase (SymRK) receptor triggers a signal transduction cascade essential for nodulation leading to cortical cell divisions, infection thread (IT) formation and final release of rhizobia to the intracellular space, forming the symbiosome. Herein, the participation of SymRK receptor during the nodule organogenesis in Phaseolus vulgaris is addressed. Our findings indicate that besides its expression in the nodule epidermis, in IT, and in uninfected cells of the infection zone, PvSymRK immunolocalizes in the root and nodule vascular system. On the other hand, knockdown expression of PvSymRK led to the formation of scarce and defective nodules, which presented alterations in both IT/symbiosome formation and vascular system.
Assuntos
Phaseolus/genética , Proteínas de Plantas/metabolismo , Nodulação , Proteínas Serina-Treonina Quinases/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Regulação para Baixo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Phaseolus/enzimologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Proteínas Serina-Treonina Quinases/genética , Nódulos Radiculares de Plantas/enzimologia , Transdução de Sinais , SimbioseRESUMO
BACKGROUND: Cooking time decreases when beans are soaked first. However, the molecular basis of this decrease remains unclear. To determine the mechanisms involved, changes in both pectic polysaccharides and cell wall enzymes were monitored during soaking. Two cultivars and one breeding line were studied. RESULTS: Soaking increased the activity of the cell wall enzymes rhamnogalacturonase, galactanase and polygalacturonase. Their activity in the cell wall was detected as changes in chemical composition of pectic polysaccharides. Rhamnose content decreased but galactose and uronic acid contents increased in the polysaccharides of soaked beans. A decrease in the average molecular weight of the pectin fraction was induced during soaking. The decrease in rhamnose and the polygalacturonase activity were associated (r = 0.933, P = 0.01, and r = 0.725, P = 0.01, respectively) with shorter cooking time after soaking. CONCLUSION: Pectic cell wall enzymes are responsible for the changes in rhamnogalacturonan I and polygalacturonan induced during soaking and constitute the biochemical factors that give bean cell walls new polysaccharide arrangements. Rhamnogalacturonan I is dispersed throughout the entire cell wall and interacts with cellulose and hemicellulose fibres, resulting in a higher rate of pectic polysaccharide thermosolubility and, therefore, a shorter cooking time.